The extraction, concentration and purification of biologically active substances (enzymes, antibodies, hormones, DNA or RNA fragments, and the like) is one of the fundamental problems in biotechnology. The discovery and synthesis of this class of substances, which generally occur with various proportions of other substances present as impurities, has stimulated the search for and development of new techniques and the improvement of known techniques with the object of achieving greater specificity and, where possible, with the greatest economy of time and resources.
These problems of preparative and fractionation techniques, especially as regards the quality (purity) of the products or isolated fractions, take on a special meaning in the case of materials or proteinaceous fractions sic!, commonly composed of various proteins having similar characteristics, often difficult to resolve, as in the case of the fractionation of organ extracts or lysates.
Moreover, this requirement, insofar as it relates to the purity of the biologically active substances and fractions, which is necessary in research work, becomes essential where medicinal products are concerned, as in the case of interferon, insulin, monoclonal antibodies, and the like.
The need to have methods of ever-increasing selectivity has led to the development of other techniques of varying complexity, for example the widely diversified chromatographic techniques such as pawl sic!, high pressure and thin-layer chromatography and, more recently, molecular exclusion chromatography and affinity chromatography, combined in practice with other techniques based on other principles, such as electrophoresis, dialysis, and the like.
Molecular exclusion chromatography is based on the size and/or geometry of the solute or to isolate sic! or purified, while ion exchange chromatography is based on the coulombic interaction between the substrate and the solute and the acid/base properties of the solute.
For the purposes of the present invention, it is appropriate to stop and consider this chromatographic variant known as "affinity chromatography" (Hay and Dean; Chem. and Ind. 1981; p. 726), based on specific non-covalent interactions between the substance of interest and a suitable reactant immobilized on a suitable substrate (also known as matrix), an interaction which obviously does not take place with the other components (or impurities) present in the sample. In distinction to the other components, the substance of interest remains bound or immobilized on the substrate. This occurs, for example, with certain enzymes (in the sample) which bind strongly but reversibly to specific coenzymes, or an antibody binds to and becomes immobilized with the specific antigen (or vice versa). The next steps are obvious: the combination or complex of substance of interest with the substrate may be processed once separated from the unbound components of the sample, the complex is subsequently split and the substance of interest is recovered, naturally with a higher degree of purity.
This property is exploited for isolating one enzyme from others (or from other solutes) by making use of affinity chromatography, binding the specific coenzyme or the cofactors (NAD, AMP) for the enzyme to be separated or purified covalently to a suitable functional group located at the surface of the particles which make up the support of the chromatographic column ("spacer arms"), and then eluting with a solution of the free ligand (coenzyme). The same principle underlies the application of other bonding species such as inhibitors, antigens, antibodies, lectins, and the like.
As a support material, certain polysaccharides such as agarose and dextrans (for example the commercial products known as Sepharose or Sephadex, and the like), or polyacrylates (for example the commercial products known as Bio-Gel, Trisacryl, Ultrogel, and the like), are commonly used. The support material is usually activated, before the operation of binding the chosen ligand, with cyanogen bromide, for example. The largest possible activator/support ratio is generally applied, with the object of achieving a high binding capacity in the chromatographic column.
Recently, the resolving power of affinity chromatography columns has been improved through the application of "spacer arms", designed to compensate for the steric hindrance features resulting from the size and geometry of biologically active molecules of interest. Said spacer arms (aliphatic groups such as, for example, polymethylene chains of 1 to 10 carbon atoms) are pendant groups which project from the surface of the particles of the support, at the free end of which the ligand groups are bound (Low, C. R: Topics in Enzyme and Fermentation Biotechnology, 5a. ed, Wiseman, A: Ellis Horwood, Chichester, 1981, chap. 2).
The resolving power of affinity chromatography has undergone an unexpected improvement as a result of the discovery of a clear affinity between dyes and biologically active molecules, especially proteins, which has led to the fractionation of these biologically active molecules by affinity chromatography, supports and/or colored matrices being employed, as disclosed, for example, in:
Eur. Pat. Sol. EP 183198 (Asahi Chemical Ind. Co. Ltd.) PA1 PCT Int. Sol. WO 84/4309 (Pharmacia AB) PA1 Eur. Pat. Sol. EP 27262 (Dupont de Nemours E.I. and Co.) PA1 USSR SU 1168564 (All Union Scientific Research Inst. of applied Microbiology) PA1 a) the disclosures of the prior art in relation to colored polymers formed by copolymerization of colorless monomers or prepolymers and colored monomers (see above), and PA1 b) the affinity of certain chromophors for proteins in general and for bioactive proteins (such as enzymes, antibodies and hormones) in particular, and to design and develop a new composition of matter which is useful for separating substances having affinity for specific dyes which form part of a polymeric structure.
There have been developed, in addition, other methods of this type (affinity chromatography) for the purification of proteins making use of more complicated processes, such as the use of two dyes, one immobilized on the matrix and the other present in the elution fluid (Ger Offen. DE 3244006), an immobilized carrier coupled to a blue dye (Affi-gel Blue) with a residue chelated with a metal (Eur. Pat. Appl. E.P. 94672 A1) or the formation of two phases by addition to the biological fluid of a Cibacron Blue-Sepharose 68 sic!/PEG 4000 complex, with a suitable buffer (PCT INT. WO 84/4309 A1).
Methods for purifying proteins solely of high molecular weight (British Patent 2053926) are also known.
Thus, the affinity of certain dyes, and of certain matrices with dye bound to them, for substances with biological activity, including proteins, peptides, hormones, enzymes, growth and transforming factors, nucleic acids and nucleotides, has been disclosed in the prior art. This affinity has been applied for the fractionation, purification, and the like, of these substances, employing columns or matrices in which the dye molecules are bound to a colorless original matrix (formed from agarose, dextrans, polyacrylates, and the like, and combinations and variants thereof), either directly or with the interposing of "spacer arms" (Lowe op. cit.). These spacer arms permit greater steric freedom in proximity to the ligand.
In addition, the preparation and application of colored polymers, composed of finely divided or micronized pigments dispersed in a polymer matrix, for industry for purely decorative purposes, in the textile industry, packaging, and the like, covering the whole range of colors of the visible spectrum, have also been disclosed in the prior art; this includes the use of fluorescent dyes, according to a recent disclosure in U.S. Pat. No. 4,016,133 of Ilyoshu et al. An important innovation in the art of preparing colored polymers has been provided by the application of conventional techniques in the polymerization of colorless prepolymers with monomers carrying at least one covalently bound chromophor group. This hence gives rise to permanently colored polymers in which the chromophor group or structure is covalently bound to the hydro-carbon skeleton of the polymer; such is the case with the polymers obtained by Winnik et al. U.S. Pat. No. 4,795,794, which deals with the dispersion polymerization of colorless vinyl monomers and vinyl monomers covalently bound to a dye. As disclosed by Winnik et al., this class of colored polymers are sic! especially useful for the development of images in electrographic printing processes (colored toner particles).
Disclosure of the Invention
It has now been found possible to combine
Chromophor is understood to mean a chemical structure bearing color, and this is the sense in which it will be used in this invention.
The chromophor is supplied by a synthetic organic dye of the anionic type, in particular those belonging to the group of reactive, acid and/or direct dyes. These chromophors are of the azo, anthraquinone, formazan, dioxazine and/or phthalocyanine type, and are generally modified, especially by the introduction of polymerizable groups and/or spacer arms.
As examples of modified chromophors or dyes, the following may be mentioned: ##STR1##
Consequently, a subject of the present invention is the provision of a composition of matter for use in the specific separation of proteins by affinity mechanisms, including affinity chromatography, which comprises a polymer in which a chromophor with affinity for the proteins to be separated forms part of the molecules.
A further subject of the present invention is the fact that the polymer is a synthetic polymer of a monomer carrying a chromophor with affinity for the proteins to be separated, said chromophor being a dye modified by the introduction of polymerizable or copolymerizable groups. Moreover, the content of the modified chromophor is not less than 0.1% by weight.
Yet a further subject consists of the feature that the modification of the chromophor includes the presence of spacer arms, in particular (C.sub.1 -C.sub.6) alkyl chains.
Yet a further subject consists of the feature that the copolymer is formed from a colored monomer and a colorless monomer, the latter being a vinyl monomer chosen from the group comprising vinyl acetate, divinylbenzene, styrene, acrylic acid, methacrylic acid, methacrylamide and the amides N-2-hydroxy-1,1-bis(hydroxymethyl)ethyl!-2-propenamide and N,N'-methylenebis(2-propenamide).
Yet a further subject consists of the feature that the degree of crosslinking of the copolymer has been regulated during its synthesis so as to obtain the pore size and other appropriate properties for the binding of the proteins of interest.
Yet a further subject of the present invention consists of the feature that the chromophor is supplied by a synthetic organic dye of the anionic type, in particular those chosen from the group composed of reactive dyes, acid dyes and direct dyes, these chromophors being chosen from the group comprising azo, anthraquinone, formazan, dioxazine and phthalocyanine dyes modified by the introduction of polymerizable and copolymerizable groups.
Yet a further subject of the present invention consists of a method for preparing the composition of matter for use in the specific separation of proteins, which comprises the polymerization of at least one monomer of which a chromophor with affinity for the proteins to be separated forms part.
In an embodiment of said method, the polymerization of at least two monomers, each of which contains a chromophor with affinity for the proteins to be separated, has been envisaged, said chromophors being different from one another.
Yet a further subject consists of the feature that each chromophor is supplied by a synthetic organic dye of the anionic type, in particular those chosen from the group composed of reactive dyes, acid dyes and direct dyes, these chromophors being chosen from the group comprising azo, anthraquinone, formazan, dioxazine and phthalocyanine dyes modified by the introduction of polymerizable and copolymerizable groups.
Another additional subject of the present invention is a method for separating proteins for sic! the composition of matter referred to above, which comprises bringing the material carrying the protein to be separated in solution into contact with said composition of matter in divided or granular form, eluting the substances selectively retained by said component and recovering the appropriate fractions.
In a special case of said method, the composition of matter is the contents of an affinity chromatography column.
A further subject of the method described above is the mixing of said composition in divided or granular form under conditions which ensure intimate contact between said composition of matter in divided or granular form and the material carrying the proteins to be separated in solution, and then the separation by filtration, decantation or centrifugation of the divided material loaded with the proteins retained by affinity by the dye fraction bound to the polymer.
Yet a further subject of the method for separating proteins with the abovementioned composition of matter is the fact that at least one of the proteins of interest to be separated is present in a small proportion compared to the other components of the mixture.